CANDIDATE/TRAINING: My goal for this career development award is to obtain the necessary training to become an independent physician-scientist studying novel circuits that control regulatory T cell (Treg) differentiation and functio. I am a transfusion medicine fellow who earned a Ph.D. in Immunology studying the regulation of T cell receptor gene assembly. Subsequently, I began pursuing a specific interest in the differentiation of na?ve T cells into TH subsets. Building on foundational skills in molecular genetics, I now plan to add expertise and mentored research experience in cellular immunology, chemical biology and animal models of inflammation to further our understanding of Treg development and function in the context of inflammatory disease. My mentor is an immunologist leader in IBD genetics and biology and provides an unparalleled environment uniquely suited to the success of this project and my career. My research advisory committee brings all necessary additional expertise and my oversight committee comprises successful investigators who are excellent mentors. My prior research and clinical training, together with mentored research and coursework during the award period will build fundamental skills and highlight important findings in the regulation of Treg differentiation that I will follow up as an independent, R01-funded investigator with the long-term goal of understanding how T cell biology is modulated in the context of inflammatory disease. PROJECT: Tregs are critical anti-inflammatory cells, impaired development and/or function of which leads to inflammatory disease. Emerging data shows that Tregs typically exhibit organ-specific specializations to facilitate niche-relevant roles, calling nto question whether current approaches to expand Tregs ex vivo can adequately reproduce such functions. I hypothesize that local microenvironments contain the cues necessary to induce specializations required for niche-specific functions. Our pioneering unbiased chemical biology efforts identified novel small molecule enhancers of Treg differentiation, including harmine and 2 scaffolds from the Broad DOS collection. The experimental pipeline described in Specific Aim 1 highlights optimized enhancers based on these scaffolds. Harmine and the optimized DOS enhancers are used to address the core hypothesis in Specific Aim 2, that delivery of small molecule Treg enhancers to mucosal surfaces promotes Treg development and function locally and distally, both at steady state and using in vivo models of inflammation. In Specific Aim 3, we address novel and testable mechanistic hypotheses raised by pilot studies with harmine, including lineage plasticity, as well as the role of novel candidate genes (DYRK1a and DYRK2) and pathways (Creb and NF-kB1 signaling) in Treg differentiation. These studies have important impact on the conceptual approach to Treg-based therapy and are likely to highlight novel regulators of Treg differentiation, as well as point to multidisciplinary approaches to rapidly generate mechanistic hypotheses, enhancing our basic understanding of Treg biology in the context of inflammatory disease.